CN114891802A - Application of OsDUF6 gene and encoding protein thereof in salt tolerance breeding of rice - Google Patents

Abstract

The invention discloses a OsDUF6 gene which is separated and cloned from rice DNA and is related to rice salt tolerance. The protein coded by the gene contains a Domain of Unknown Function (DUF), has a transmembrane Domain and is a membrane and nuclear double localization protein. The salt tolerance and the anti-oxidative stress capability of the knockout mutant of OsDUF6 are reduced, which shows that the knockout mutant is used for positively regulating the salt tolerance and the anti-oxidative stress capability of rice and can be applied to improving the salt tolerance of cultivated rice.

Description

Application of OsDUF6 gene and encoding protein thereof in salt tolerance breeding of rice

Technical Field

The invention belongs to the field of genetic engineering, and relates to a gene related to rice production and stress resistance. In particular to application of OsDUF6 gene and coded protein thereof in rice salt tolerance breeding.

Background

Rice is the most important grain crop in China and even all over the world, provides grain for more than 50% of the population all over the world, and is also the most important grain in China. Therefore, how to improve the yield and the stress resistance of the rice under the situation of global environmental change is an effective means for guaranteeing the food safety of China.

On one hand, the problem of soil salinization of the paddy field is more and more serious, so that the normal growth of the paddy rice is seriously threatened, and the yield is reduced; on the other hand, in order to meet the continuously expanded food demand of China and guarantee the food safety of China, rice planting needs to be expanded to saline-alkali soil in Xinjiang and coastal mudflats in southeast. Therefore, improvement of the salt tolerance of rice is becoming more and more important.

Therefore, it is necessary to discover, research and apply the salt-tolerant gene resources of rice and apply the salt-tolerant gene resources to the improvement and breeding of the salt-tolerant performance of rice.

Disclosure of Invention

Based on the above, the invention aims to provide an OsDUF6 gene and application of a protein coded by the gene in salt tolerance breeding of rice. The specific technical scheme is as follows:

an application of OsDUF6 gene in regulation and control of rice salt tolerance, wherein the OsDUF6 gene comprises:

a DNA segment with a nucleotide sequence shown as SEQ ID NO. 3; or

A DNA fragment at least 90% homologous to SEQ ID NO 3; or

A subfragment functionally corresponding to the nucleotide sequence shown in SEQ ID NO. 3.

In some embodiments, the application is the application of the protein coded by the OsDUF6 gene in regulating and controlling the salt tolerance of rice.

In some embodiments, the amino acid sequence of the protein encoded by the OsDUF6 gene is shown in SEQ ID NO. 4; or

The amino acid sequence of the protein coded by the OsDUF6 gene is selected from one of homologous sequence, conservative variant, allelic variant, natural mutant and induced mutant of SEQ ID NO. 4.

The invention also provides an application of the gene or the protein-related biomaterial in regulation and control of rice salt tolerance, wherein the biomaterial is any one of the following:

a) an expression cassette containing a DNA fragment with a nucleotide sequence shown as SEQ ID NO. 3, or an expression cassette containing a DNA fragment which is at least 90% homologous with SEQ ID NO. 3;

b) a recombinant vector containing a DNA fragment with a nucleotide sequence shown as SEQ ID NO. 3 or a recombinant vector containing a DNA fragment which is at least 90% homologous with SEQ ID NO. 3; preferably, the recombinant vector is a knock-out vector, a knock-down vector or an overexpression vector;

c) a recombinant microorganism, a recombinant cell, a transgenic plant tissue or a transgenic plant containing the recombinant vector of b);

d) tissue culture or protoplast of regenerable cells of the transgenic plant of c).

In some embodiments, the plasmid used to construct the recombinant vector is a Ti plasmid or a plant viral vector.

The invention also provides an OsDUF6 gene knockout mutant based on the CRISPR/Cas9 technology, and the OsDUF6 gene knockout mutant is prepared by the following steps:

s1, designing 2 sgRNAs and 2 pairs of target sequence primers, wherein the sgRNAs are designed according to a nucleotide sequence shown in SEQ ID NO. 5, and the nucleotide sequence of the target sequence primers is shown in SEQ ID NO. 6-9;

s2, constructing a sgRNA expression cassette by side-cutting ligation and overlapping extension PCR, inserting a linear pYLCISPR/Cas 9Pubi-H vector, and carrying out agrobacterium transformation and screening to obtain the recombinant plasmid.

In some embodiments, the OsDUF6 knockout mutant is characterized in that the nucleotide sequence of the primer used in the overlap extension PCR is shown as SEQ ID NO. 6-13.

The present invention also provides a method of breeding rice having salt tolerance by selecting rice comprising the OsDUF6 gene according to claim 1.

In some of these embodiments, the breeding method is: constructing the recombinant vector and culturing the transgenic rice containing the recombinant vector, wherein the recombinant vector is an overexpression vector.

In some embodiments, the transgenic rice overexpresses a protein encoded by the OsDUF6 gene.

The invention provides an OsDUF6 gene and a DUF protein coded by the same, which have a typical DUF protein family structural domain and a transmembrane structural domain, and the salt tolerance of rice seeds at the germination stage and the seedling stage can be influenced by knocking out or knocking down the DUF6 gene.

The invention separates a cloned DNA fragment of a complete coding segment from rice, and analyzes the protein sequence coded by the gene to show that the DNA fragment codes DUF family protein and has a transmembrane structural domain, so the gene is named as OsDUF 6.

The gene or homologous gene of the present invention is obtained by screening cDNA library and genomic library using the cloned OsDUF6 gene as a probe. The OsDUF6 gene of the invention and any DNA with homology of more than 90% can also be obtained by amplifying from genome, mRNA and cDNA by using PCR (polymerase chain reaction) technology. Any vector designed according to the gene sequence of OsDUF6 and capable of guiding exogenous DNA to knock out (knock out) or knock down (knock down) OsDUF6 in plants, and transgenic plants with reduced salt tolerance can be obtained by transforming plants after the vector is connected.

The recombinant (knockout or knock-down) vector carrying the OsDUF6 gene can be introduced into plant cells by using Ti plasmids and plant virus vectors and by using conventional biotechnology methods such as direct DNA transformation, microinjection and electroporation.

The transformation host of the OsDUF6 gene knockout mutant is rice.

The invention provides a rice DNA fragment containing an 297bp coding gene OsDUF6 by separating, cloning and knocking down rice genes. The gene contains domains typical of the DUF family of proteins. The OsDUF6 gene is related to salt tolerance of rice.

The invention can be used for researching a molecular method for obtaining transgenic plants by genetic transformation of the gene.

The rice gene of the invention has obvious influence on plant height, and can be applied to rice salt tolerance improvement breeding.

The salt tolerance of the OsDUF6 functional deletion mutant seed in the germination stage and the seedling stage is obviously reduced by using the CRISPR-Cas9 technology. Therefore, the OsDUF6 gene is a positive regulation factor of rice salt tolerance and has a wide application prospect in rice breeding.

Drawings

FIG. 1 shows the result of comparison of the predicted protein sequence of OsDUF6 (Gene No.: Os06g0269200) gene with the homologous protein sequence using ClustalW2 software according to the present invention.

FIG. 2 shows that the present invention uses TMHMM Serverv.2.0 to predict the transmembrane domain of OsDUF6 protein, and the OsDUF6 protein is found to have 3 transmembrane domains.

FIG. 3 shows the changes of gene sequence (a) and protein sequence (b) of a knockout mutant created by the OsDUF6 gene based on the Crisper-Cas9 technology.

FIG. 4 is the result of subcellular localization of OsDUF6 gene of the present invention, showing membrane and nucleus double localization.

FIG. 5 shows the result of the decrease in salt tolerance of the OsDUF6 knockout mutant of the present invention relative to the wild type at the seed germination stage.

FIG. 6 shows that the seedling-stage salt tolerance of the OsDUF6 gene knockout mutant is remarkably reduced as compared with the wild type seedling-stage drought tolerance.

Detailed Description

In order that the invention may be more readily understood, reference will now be made to the following more particular description of the invention, examples of which are set forth below. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. The examples are given solely for the purpose of illustration and are not intended to be limiting. It is intended that all modifications or alterations to the methods, procedures or conditions of the present invention be made without departing from the spirit or essential attributes thereof. These embodiments are provided so that this disclosure will be thorough and complete.

It is understood that the experimental procedures, for which specific conditions are not indicated in the following examples, are generally performed according to conventional conditions, such as molecular cloning in Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. The reagents used in the examples were commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The invention is further illustrated and described in detail by the following examples:

example 1 cloning of OsDUF6 Gene of Rice

1. Seedling cultivation

The rice variety Nipponbare is put at 30 ℃ for germination for 48 hours, then the rice variety Nipponbare is sowed in a greenhouse, and when the number of rice leaves is 3-5, DNA or RNA is prepared to be extracted.

RNA isolation:

extraction of RNA: freezing the sample in a mortar by using liquid nitrogen, grinding the sample into powder, adding a 2mL EP tube containing 1mL TRNzol-A + reagent, fully oscillating, standing at room temperature for 5min, adding 0.2mL chloroform, violently oscillating for 15s, and standing at room temperature for 3 min; after centrifugation at 12000rpm for 10min at 4 deg.C, the supernatant was transferred to a new 2mL EP tube, an equal volume of isopropanol was added to precipitate RNA, and 100. mu.L of RNase-free ddH was added ₂ And dissolving the O. The total RNA quality is identified by electrophoresis, and then the RNA content is determined on a spectrophotometer.

3. Reverse transcription to synthesize first strand cDNA

(1) The extracted RNA sample was digested with DNaseI before reverse transcription in the following reaction scheme:

after 15min at 37 ℃ the reaction was stopped by adding 0.25. mu.L of 0.1M EDTA (to ensure a final concentration >2mM), incubating at 70 ℃ for 10min, and briefly centrifuged and placed on ice for further use.

(2) First strand cDNA was synthesized according to the Promega reverse transcription System A3500 handbook, with the following steps:

the following reagents were added to the DNaseI digested sample in order to prepare a 20. mu.L reaction system:

incubating the reaction system at 42 ℃ for 15 min; then heating at 95 deg.C for 5min to inactivate AMV reverse transcriptase and prevent it from binding to DNA; standing at 4 deg.C or on ice for 5 min. The prepared cDNA can be used immediately or stored at-20 ℃ for use.

4. Amplification of coding region (CDS) of OsDUF6 gene of rice

The coding region (CDS) sequence of rice OsDUF6 (gene number: Os06g0269200) was obtained by searching the rice genome and the full-length gene database. And designing PCR amplification primers according to the prediction information. The primer sequence is as follows:

OsDUF6-F ATGAAGTTATTTGGTGTGATGA(SEQ ID NO:1)

OsDUF6-R TCAACTGTCCCCTCTAGATACA(SEQ ID NO:2)

directly cloning from cDNA to obtain CDS of OsDUF6 gene, recovering gel, connecting to pEASY-Blunt vector, sequence determination after identification, and alignment confirmation of sequencing result and BLAST. The results show that the length of the full-length CDS of the rice OsDUF6 in the invention is 294bp, and the CDS is shown in SEQ ID NO. 3.

ATGAAGTTATTTGGTGTGATGATACAGTTTCCCTGGTTGTCAATACTATACACAGGCATATTCTCAACTACCTTTTGTTTATGGGCAGAGGTGGCTGCTATGCGTGATGTATCAGCTACGGAAACTGCAATAATTTATGGTCTGGAACCTGTATGGGGTGCAGCTTTTGCATGGGCTATGCTTGGTGAGAGATGGGGCATGACAGGATTTGTTGGAGCTATCTTCATCATAGCTGGTAGCTTTATGGTTCAGATACTGGGATCATTTCCTGATGTATCTAGAGGGGACAGTTGA(SEQ ID NO:3)

Example 2 protein sequence information and homology analysis of OsDUF6 in Rice

The amino acid sequence of rice OsDUF6 is deduced according to the ORF of the novel rice OsDUF6 (gene number: Os06g0269200), 97 amino acids in total and the molecular weight is 10.63K daltons, and the detailed sequence is shown in SEQ ID NO. 4 as follows:

MKLFGVMIQFPWLSILYTGIFSTTFCLWAEVAAMRDVSATETAIIYGLEPVWGAAFAWAMLGERWGMTGFVGAIFIIAGSFMVQILGSFPDVSRGDS*

through the comparison of BLASTP programs (https:// blast.ncbi.nlm.nih.gov/blast.cgi) of NCBI websites, the OsDUF6 is found to belong to a Domain of Unknown Function (DUF) family protein; the transmembrane structure of OsDUF6 was predicted by TMHMM Serverv.2.0(http:// www.cbs.dtu.dk/services/TMHMM /), and the protein OsDUF6 was found to have a transmembrane domain.

By performing multiple sequence alignment on part of DUF proteins in plants, we find that the proteins all contain a conserved DUF domain (FIG. 1); also, according to the prediction of tmhmservervv.2.0, OsDUF6 has 3 transmembrane domains (fig. 2).

Example 3 creation of OsDUF6 knock-out mutant of Rice Gene

1. Constructing a multi-target knockout vector containing OsDUF6 by using a CRISPR-Cas9 technology:

(1) guide RNA target sequence selection and primer design

2 sgRNAs were designed based on the genomic sequence of OsDUF6 (Gene No.: Os06g 0269200). The sgRNA target sequence of 20nt nucleotide follows 5' -GN ₁₉ NGG-3 'sequence (SEQ ID NO:5) is designed, target sequence primers gRT06g0269200 and OsU6aT06g0269200 are designed, 15-17nt aT 3' end are respectively matched with sgRNA and U6a promoters. And (3) comparing the designed sgRNA target sequences with a rice genome database to eliminate non-specific target cutting sites, wherein specific target nucleotide sequences are as follows. 6 of SEQ ID NO:

gRT06g0269200-1:5’-GGCTATGCTTGGTGAGAGAgttttagagctagaaat-3’

SEQ ID NO:7

OsU6aT06g0269200-1:5’-TCTCTCACCAAGCATAGCCCggcagccaagccagca-3’

SEQ ID NO:8

gRT06g0269200-2:5’-GTGAGAGATGGGGCATGACgttttagagctagaaat-3’

SEQ ID NO:9

OsU6aT06g0269200-2:5’-GTCATGCCCCATCTCTCACCggcagccaagccagca-3’

(2) performing denaturation annealing on the joint primer, and diluting the primer to the working concentration of 10umol for later use;

reaction system: 1ul gRT06g0269200-1(gRT06g0269200-2) primer +1ul gRT06g0269200-2(OsU6aT06g0269200-2) primer +8ul H ₂ O

Reaction conditions are as follows: and (3) at 90 ℃ for 30s, and then naturally cooling.

Two pairs of primers were performed separately.

(3) Edge cutting is carried out and a reaction system is connected:

reaction conditions are as follows: 5min at 37 ℃ and 5min at 20 ℃ for 5 cycles

Two pairs of primers were performed separately.

(4) The ligation product was cut at the same time as a template, and a first round of PCR was performed. KOD NEO-plus from Toyobo

This PCR product was designated u1

This PCR product was named g1

The reaction conditions are as follows: heating at 98 deg.C for 3 min; 15s at 98 ℃; at 58 ℃ for 20 s; 68 ℃ for 20 s; at 68 ℃ for 2 min; 12 ℃ for 10 min. 30 cycles.

The second target repeats the above experiment, and the primer pairing is noticed (U-F +)

OsU6aT06g 0269200; gRT06g0269200+ gRNA-R) was named u2, g 2.

The primer sequence is as follows:

U-F:5’-CTCCGTTTTACCTGTGGAATCG-3’(SEQ ID NO:10)

gR-R:5’-CGGAGGAAAATTCCATCCAC-3’(SEQ ID NO:11)

(5) overlap extension, step 4 as template, using KOD NEO-plus, also from Toyobo

2ul u1+2ul g1+16ul ddH are taken ₂ O mix (for the purpose of 10-fold dilution of the product from step 4) and named u1+ g1

This PCR product was named 6-T1

The second target repeats the above experiment with the template u2+ g2

Reaction conditions are as follows: as above.

This PCR product was named 6-T2

The primer sequences required in the reaction system are as follows:

U-GAL:5’-ACCGGTAAGGCGCGCCGTAGTGCTCGACTAGTATGGAATC

GGCAGCAAAGG-3’(SEQ ID NO:12)

Pgs-GAR:5’-TAGCTCGAGAGGCGCGCCAATGATACCGACGCGTATCCA

TCCACTCCAAGCTCTTG-3’(SEQ ID NO:13)

(6) purification of the overlap extension product (using 3mol/L sodium acetate, pH5.2)

20ul overlap extension product +70ul ddH ₂ O +10ul 3M sodium acetate, mixing well and adding 200ul Ice absolute ethanol (absolute ethanol to-20 deg.C low temperature storage for a period of time, centrifugation to remove supernatant, 75% ethanol washing once, centrifugation to remove supernatant, air drying, adding ddH ₂ O 15ul。

(7) The edge trimming and finishing carrier comprises the following systems:

enzyme digestion at 37 deg.C for 10min

Then is added

T4 DNA ligase buffer (NEB) 0.5ul

T4 DNA ligase (NEB) 0.1ul

Reaction conditions are as follows: 2min at 37 ℃, 3min at 10 ℃, 5min at 20 ℃ and 12-15 cycles.

(8) And (4) directly carrying out transformation after the completion, coating an LB (Langmuir-Blodgett) plate containing kanamycin to screen positive clones, and picking positive single clones the next day to carry out sequencing verification.

2. Agrobacterium transformation

(1) Preparation of agrobacterium tumefaciens (EHA105) competent cells:

culturing Agrobacterium tumefaciens bacterial solution at 28 deg.C until OD600 is 0.5, centrifuging at 4 deg.C, collecting thallus, and ice-cooling with CaCl 500 μ L and 0.1mol/L ₂ Resuspending, ice-cooling for 30min, centrifuging, removing supernatant, and washing with 100. mu.L of 0.1mol/L ice CaC1 ₂ After resuspension, it was stored at 4 ℃.

(2) Agrobacterium transformation, adopting a freeze-thaw method:

adding 5 μ L plant expression vector plasmid DNA into Agrobacterium infection state cell (100 μ L), mixing, ice-water bath for 30min, and rapidly freezing and quenching in liquid nitrogen for 2 min; adding 400-800. mu.L YEP culture solution (containing kanamycin and Kan); carrying out shaking culture at 28 ℃ and 200r/min for 3-5 h; centrifuging at room temperature (5000r/min, 5min), keeping 100 μ L of supernatant, resuspending thallus, coating on LB solid culture medium (containing Kan), performing inverted culture at 28 deg.C for 2 days until a colony of appropriate size grows out, and selecting single clone for PCR detection to obtain positive strain.

3. Callus induction: rinsing the seeds with sterile water for 15-20min, sterilizing with 75% ethanol for 1min, and sterilizing with 1.5% sodium hypochlorite solution with effective concentration for 20 min. Finally, the mixture is washed with sterile water for 5 times. The washed seeds are inoculated in the induction callus culture medium by blotting the seeds with absorbent paper, and the seeds are cultured in the dark for 2 weeks at 25 ℃.

Callus induction medium: the induction medium shown in Table 1 was added with proline 0.3g, casein hydrolysate 0.6g, sucrose 30g and 2.5mL of 2,4-D (concentration 1mg/mL) to prepare a 1L solution, the pH was adjusted to 5.9, agar powder 7g was added, and the solution was sterilized at high temperature and high pressure.

4. Subculturing: the embryogenic callus was excised, inoculated into a subculture medium, and cultured in the dark at 25 ℃ for 2 weeks.

Subculture medium: adopting the subculture medium shown in Table 1, adding 0.5g of proline, 0.6g of hydrolyzed casein protease, 30g of sucrose and 2mL of 2,4-D (concentration 1mg/mL) to prepare 1L solution, adjusting pH to 5.9, adding 7g of agar powder, and sterilizing at high temperature and high pressure.

5. Agrobacteria dip dyeing and callus co-culture: culturing agrobacterium, selecting positive single colony, culturing in 1mL agrobacterium culture solution (containing antibiotic) at 28 ℃ overnight; the above culture was added to 50mL of Agrobacterium culture medium (containing antibiotics) and cultured at 28 ℃ until OD600 became 0.6-1.0. And centrifuging the obtained agrobacterium liquid, adding the collected thalli into a suspension culture solution, and performing shake culture for 30min until OD600 is 0.6-1.0. Then placing the callus into suspension culture solution containing agrobacterium liquid, and carrying out shake culture for about 20 min. Air drying the callus on sterilized filter paper, transferring into co-culture medium, and dark culturing at 25 deg.C for 5 d.

Suspension culture solution: 0.08g of hydrolyzed casein, 2g of sucrose and 0.2mL of 2,4-D (concentration: 1mg/mL) were added to the suspension culture medium shown in Table 1 to prepare 100mL of a solution, the pH was adjusted to 5.4, the solution was divided into two bottles (50 mL each), and the solution was sterilized by autoclaving at high temperature. 1mL of 50% glucose and 100. mu.L of AS (100mM) were added prior to use.

Co-culture medium: the co-culture medium shown in Table 1 was used, and 0.8g of hydrolyzed casein protease, 20g of sucrose and 3.0mL of 2,4-D (concentration: 1mg/mL) were added to prepare 1L of a solution, the pH was adjusted to 5.6, 7g of agar powder was added, and high-temperature autoclaving was performed. 20mL of 50% glucose and 1mL of AS (100mM) were added prior to use.

6. Screening and culturing: after co-culturing for 3 days, selecting the good callus, transferring the callus into a screening culture medium, carrying out dark culture at 25 ℃ for 2 weeks, and screening twice.

Screening a culture medium: using the selection medium shown in Table 2, 0.6g of hydrolyzed casein protease, 30g of sucrose and 2.5mL of 2,4-D (concentration: 1mg/mL) were added to prepare 1L of a solution, the pH was adjusted to 6.0, 7g of agar powder was added, and the solution was sterilized by autoclaving at high temperature. 1mL Hn and 1mL Cn (100ppm) were added prior to use.

7. Differentiation culture: selecting embryogenic callus, inoculating into differentiation culture medium, culturing at 24 deg.C for 16h/8h in light and dark to induce differentiation bud (4-6 weeks).

Differentiation medium: adopting the differentiation culture medium shown in Table 2, adding 2.0 mg/L6-BA, 2.0mg/L KT, 0.2mg/L NAA, 0.2mg/L IAA, 1.0g of hydrolytic casein and 30g of sucrose to prepare 1L solution, adjusting pH to 6.0, adding 7g of agar powder, and sterilizing at high temperature and high pressure.

8. Rooting culture: when the bud grows to about 2cm, cutting off the bud, inserting the bud into a rooting culture medium, culturing at about 25 ℃ in 16h/8h in light and dark, and inducing to root.

Rooting culture medium: the rooting medium shown in Table 2 was added with 30g of sucrose to prepare 1L of solution, the pH was adjusted to 5.8, 7g of agar powder was added, and the solution was sterilized at high temperature and high pressure.

9. Culturing a transformed plant: opening the test tube mouth after the root system is developed, adding sterile water to harden the seedlings for 2-3d, taking out the plants, washing the attached solid culture medium with sterile water, transferring the solid culture medium into soil, shading and avoiding wind at the beginning, and performing conventional field or greenhouse management culture after the plants are robust.

TABLE 1 minimal Medium composition 1

TABLE 2 minimal Medium composition 2

10. Detection of knockout mutant plant positive strains

(1) Extracting genome DNA: soaking the leaves of the sample to be tested in liquid nitrogen, grinding into fine powder, putting into a10 mL centrifuge tube, adding 4mL of 1.5 xCTAB preheated at 56 ℃, and uniformly mixing; quickly placing in 56 deg.C water bath for 30min, and reversing for several times; adding chloroform/isoamyl alcohol (24:1)4mL, and shaking gently for 30 min; centrifuging at 4000rpm for 20min, sucking 3mL of the supernatant into a new centrifuge tube (10mL), adding 300. mu.L of 10% CTAB (preheated in a 56 ℃ water bath), and 3.3mL of chloroform/isoamyl alcohol (24:1), and inverting several times; centrifuging at 4000rpm for 20min, sucking 2.7mL of supernatant into a new centrifuge tube (10mL), adding 5.4mL of 1% CTAB (preheating at 56 ℃), gently shaking to precipitate DNA, centrifuging at 4000rpm for 20min, discarding supernatant, adding 2mL of 1M NaCl solution containing 1 muL of RNase, dissolving in 56 ℃ water bath overnight, adding 2 times volume of precooled (-20 ℃) absolute ethanol to precipitate DNA, centrifuging at 4000rpm for 5min, discarding supernatant, washing precipitate with 75% ethanol, air drying, and adding 100 muL of sterilized water to dissolve DNA.

(2) PCR amplification and clone sequencing of the knock-out mutant OsDUF6 gene: the same as in examples 1-4.

The results show that: aiming at the sequence analysis of 3 knock-out mutant OsDUF6 genes, we find that the CDS sequence of the OsDUF6-1 knock-out mutant has deletion of A base at 199; osduf6-2 has a deletion of the T base at position 200; osduf6-3 has a T base insertion at position 200 (FIG. 3 a). Amino acid sequence analysis showed that mutations at these bases resulted in premature termination of the OsDUF6 protein, rendering the OsDUF6-1/-2/-3 protein truncated with amino acid residues, respectively (fig. 3 b). The above results indicate that the OsDUF6 gene knockout was successful in the above mutants.

Example 4 subcellular localization of OsDUF6

(1) The same procedure as in example 1 was repeated for the extraction of rice RNA and the inversion of cDNA.

(2) Design of primers OSDUF6-F and OSDUF6-R for the Gene sequence of OSDUF6

OSDUF6-F cagtGGTCTCacaacATGAAGTTATTTGGTGTGAT(SEQ ID NO:14)

OSDUF6-R cagtGGTCTCatacaACTGTCCCCTCTAGATACAT(SEQ ID NO:15)

(3) Using the cDNA in the step 1 as a template, amplifying a cDNA fragment of OsDUF6, connecting the cDNA fragment to a pAN580 vector, fusing OsDUF6 with GFP, transiently expressing an OsDUF6-GFP expression vector and a membrane marker protein GmAOX1-RFP in tobacco leaf protoplasts, and observing fluorescence by using a laser confocal microscope (FV 10). .

The results show that: the green fluorescent protein OsDUF6 was mostly coincident with the membrane system labeled red fluorescence, and in addition, there was a clear signal in the nucleus (fig. 4), indicating that OsDUF6 is a protein localized to both the membrane system and the nucleus.

Example 5 identification of salt tolerance of knockout mutant Material in seed Germination stage

The germination rate of seeds under the stress condition can reflect the stress tolerance of plants, so that the germination rates of the mutant and the wild type under the water condition and the salt stress condition are measured to test the effect of the gene on the salt tolerance of rice. Selecting 30 full rice seeds, putting the rice seeds into a glass culture dish with the diameter of 7cm and paved with filter paper, and adding 12ml of ddH ₂ O (as control, CK) or 200mM NaCl (salt treated), was placed in a 25 ℃ incubator (GXZ type multi-stage Programming, Ningbo south Instrument works) for dark culture. The germination rate is recorded for 1 time every 24 hours until the germination rate is 100 percent or the germination rate does not rise any more for 3 continuous days. Each material was replicated 3 times, each replication being 1 petri dish. The germination rate is the number of germinated seeds/total number of seeds × 100%.

The results show that: under control hydroponic conditions, mutants (osduf6-1/2/3) germinated slightly later than wild type, but the final germination rate was equal to that of wild type (fig. 5 a); under the salt treatment conditions, the final germination rate of the mutant is significantly lower than that of the wild type (fig. 5b), indicating that the knockout of the OsDUF6 reduces the salt tolerance of the rice seeds at the germination stage.

Example 6 identification of salt tolerance of knockout mutant materials at seedling stage

The seeds of the knock-out mutant family are hulled and disinfected (75% alcohol treatment for 1min, 1.5% NaClO treatment for 20min, and sterile water washing for 5 times). Then soaking the seeds in clear water for 24h, accelerating germination for 48 h at 37 ℃, selecting the seeds with good germination and consistent growth vigor, transferring the seeds to a 96-well plate, culturing the seeds by using a conventional nutrient solution of rice, and changing the nutrient solution every 3 to 5 days. After 20 days of growth, the plants were grown in the 4-leaf stage, and then were subjected to salt stress treatment with 180mM NaCl, and were rehydrated 5 days after NaCl treatment, and the survival rate was measured 3 days after rehydration. The experiment was set up in 3 replicates, each replicate with 48 individuals.

The nutrient solution ingredients are as follows:

the results show that: the survival of the knockout mutant of OsUDF6 after 5 days of 180mM NaCl treatment was significantly lower than that of the wild type (fig. 6). This indicates that the OsUDF6 knockout can reduce the salt tolerance of the mutant in the seedling stage and positively regulate the salt tolerance of the rice.

The technical features of the above embodiments can be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the above embodiments are not described, however, as long as there is no contradiction between the combinations of the technical features, the scope of the present description should be considered as being described in the present specification.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

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ttctcaacta ccttttgttt atgggcagag gtggctgcta tgcgtgatgt atcagctacg 120

gaaactgcaa taatttatgg tctggaacct gtatggggtg cagcttttgc atgggctatg 180

cttggtgaga gatggggcat gacaggattt gttggagcta tcttcatcat agctggtagc 240

tttatggttc agatactggg atcatttcct gatgtatcta gaggggacag ttga 294

<210> 4

<211> 97

<212> PRT

<213> Oryza sativa

<400> 4

Met Lys Leu Phe Gly Val Met Ile Gln Phe Pro Trp Leu Ser Ile Leu

1 5 10 15

Tyr Thr Gly Ile Phe Ser Thr Thr Phe Cys Leu Trp Ala Glu Val Ala

20 25 30

Ala Met Arg Asp Val Ser Ala Thr Glu Thr Ala Ile Ile Tyr Gly Leu

35 40 45

Glu Pro Val Trp Gly Ala Ala Phe Ala Trp Ala Met Leu Gly Glu Arg

50 55 60

Trp Gly Met Thr Gly Phe Val Gly Ala Ile Phe Ile Ile Ala Gly Ser

65 70 75 80

Phe Met Val Gln Ile Leu Gly Ser Phe Pro Asp Val Ser Arg Gly Asp

85 90 95

Ser

<210> 5

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gnnnnnnnnn nnnnnnnnnn ngg 23

<210> 6

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ggctatgctt ggtgagagag ttttagagct agaaat 36

<210> 7

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

tctctcacca agcatagccc ggcagccaag ccagca 36

<210> 8

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gtgagagatg gggcatgacg ttttagagct agaaat 36

<210> 9

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gtcatgcccc atctctcacc ggcagccaag ccagca 36

<210> 10

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ctccgtttta cctgtggaat cg 22

<210> 11

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

cggaggaaaa ttccatccac 20

<210> 12

<211> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

accggtaagg cgcgccgtag tgctcgacta gtatggaatc ggcagcaaag g 51

<210> 13

<211> 56

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

tagctcgaga ggcgcgccaa tgataccgac gcgtatccat ccactccaag ctcttg 56

<210> 14

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

cagtggtctc acaacatgaa gttatttggt gtgat 35

<210> 15

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

cagtggtctc atacaactgt cccctctaga tacat 35

Claims (10)

1. The application of the OsDUF6 gene in the regulation of rice salt tolerance is characterized in that the OsDUF6 gene comprises:

   a DNA segment with a nucleotide sequence shown as SEQ ID NO. 3; or

   A DNA fragment at least 90% homologous to SEQ ID NO 3; or

   A subfragment functionally corresponding to the nucleotide sequence shown in SEQ ID NO. 3.
2. 2. The use of a protein encoded by the OsDUF6 gene of claim 1 for regulating rice salt tolerance.
3. The application of the protein coded by the OsDUF6 gene in the regulation of the salt tolerance of rice is characterized in that the amino acid sequence of the protein coded by the OsDUF6 gene is shown as SEQ ID NO. 4; or

   The amino acid sequence of the protein coded by the OsDUF6 gene is selected from one of homologous sequence, conservative variant, allelic variant, natural mutant and induced mutant of SEQ ID NO. 4.
4. 4. Use of a biological material related to the gene of claim 1 or the protein of claims 2-3 for modulating salt tolerance in rice, wherein the biological material is represented by any one of the following:

   a) an expression cassette containing a DNA fragment with a nucleotide sequence shown as SEQ ID NO. 3, or an expression cassette containing a DNA fragment which is at least 90% homologous with SEQ ID NO. 3;

   b) a recombinant vector containing a DNA fragment with a nucleotide sequence shown as SEQ ID NO. 3 or a recombinant vector containing a DNA fragment which is at least 90% homologous with SEQ ID NO. 3; preferably, the recombinant vector is a knock-out vector, a knock-down vector or an overexpression vector;

   c) a recombinant microorganism, a recombinant cell, a transgenic plant tissue or a transgenic plant containing the recombinant vector of b);

   d) tissue culture or protoplast of regenerable cells of the transgenic plant of c).
5. 5. The use of the biomaterial according to claim 4 for regulating and controlling rice salt tolerance, wherein the plasmid used for constructing the recombinant vector is Ti plasmid or plant virus vector.
6. 6. An OsDUF6 knockout mutant based on CRISPR/Cas9 technology, which is characterized in that the OsDUF6 knockout mutant is prepared by the following steps:

   s1, designing 2 sgRNAs and 2 pairs of target sequence primers, wherein the sgRNAs are designed according to a nucleotide sequence shown in SEQ ID NO. 5, and the nucleotide sequence of the target sequence primers is shown in SEQ ID NO. 6-9;

   s2, constructing a sgRNA expression cassette by side-cutting ligation and overlapping extension PCR, inserting a linear pYLCISPR/Cas 9Pubi-H vector, and carrying out agrobacterium transformation and screening to obtain the recombinant plasmid.
7. 7. The OsDUF6 knockout mutant according to claim 6, wherein the nucleotide sequence of the primer used in the overlap extension PCR is shown as SEQ ID NO. 6-13.
8. 8. A method of breeding rice having salt tolerance, which comprises selecting rice comprising the OsDUF6 gene according to claim 1.
9. 9. A breeding method of rice with salt tolerance, characterized in that the recombinant vector of claim 4 is constructed and transgenic rice containing the recombinant vector is cultivated, and the recombinant vector is an overexpression vector.
10. 10. A method as claimed in claim 9, wherein the transgenic rice overexpresses the protein encoded by the OsDUF6 gene as claimed in claim 2 or 3.

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